Abstract Nonclassical patrolling monocytes (identified as Ly6Clo in mice, and CD14dimCD16+ in humans) are distinct from classical monocytes (Ly6Chi in mice, and CD14+CD16? in humans), and exhibit a unique ability to actively patrol the vascular endothelium under both homeostatic and inflammatory conditions. Nonclassical monocytes function to remove pathogens, oxidized lipoproteins, and debris from the vasculature. The focus of this application is to identify new transcription factors that control nonclassical monocyte development. Targeting such transcription factors could allow us to increase numbers or functions of these anti-inflammatory monocytes in vivo. Enhancers regulate cell-specific patterns of gene expression and can be quantified genome-wide using ChIP-Seq directed against the histone modifications H3K4me2 and H3K27ac. Using antibodies to H3K27ac to mark active enhancer regions, we identified a large enhancer region upstream of the transcription factor Nr4a1 that shows increased activity selectively in Ly6Clo nonclassical monocytes. We chose 3 active ~1.5kb regions that were located within this large enhancer that were also conserved in human CD14dim monocytes, and we generated knockout mice for these 3 enhancers using CRISPR/Cas9 approaches in vivo. Deletion of one of these enhancer regions in mice in vivo resulted in generation of a mouse that completely lacks nonclassical Ly6Clo monocytes in blood. In Specific Aim 1, we will study this enhancer region to identify new transcription factors that control nonclassical monocyte development through regulation of Nr4a1. Specific Aim 2 will test whether these nonclassical monocytes are atheroprotective, and we will mechanistically identify the functions of this monocyte subset in early atherogenesis. The results of these studies will enhance our understanding of the roles of nonclassical monocytes in atherosclerosis, and will identify new transcription factors that control development of this anti-inflammatory monocyte subset. We anticipate that therapies that increase Nr4a1 expression and nonclassical monocyte numbers may be useful in humans to target resolution of inflammation in chronic diseases, including atherosclerosis.